Use of a Simplified Single-site PCR to Facilitate Cloning of Genomic ...

Use of a Simplif ied Single-site PCR to Facilitate

Cloning of Genomic DNA Sequences Flanking a

Transgene Integration Site Grant R. MacGregor and Paul A. Overbeek

Department of Cell Biology, Institute for Molecular Genetics and Howard Hughes Medical Institute, Baylor College of Medicine,

Houston, Texas 77030

We have used a simplified single- site PCR based strategy to isolate genomic DNA flanking the transgenic insert in a line of transgenic mice. The flanking sequences, which were refractory to more con- ventional cloning techniques, were isolated and characterized within 1 week. This strategy begins with the annealing and ligation of a single- stranded oligonucleotide to recessed 5 ' ends of restriction endonu-clease-digested, size- selected transgenic DNA. Following denaturation, a second oligonucleotide is used to prime DNA synthesis from a position within the transgenic sequences to the end of the genomic restriction fragment, finally synthesizing a complement of the ligated oligonucleotide sequence. Sub- sequently, a PCR is initiated which uses primers specific for the transgenic DNA and the newly synthesized DNA complementary to the ligated oligonucleotide. The only prerequisite data are sequence information for the transgenic DNA and Southern information regarding the size(s) of restriction fragments that contain the flanking genomic material. This report demonstrates one utility of this technique--enabling rapid isolation of specific mammalian genomic DNA sequences.

R a n d o m integration of exogenous DNA into the genome of fertilized mouse oocytes can lead to insertional inactivation of endogenous genes. 11-31 In cases where interesting phenotypes are generated, the isolation of genomic sequences flanking the site of the integrated transgenic complex provides a starting point from which to identify the inactivated gene. Often, this has been accomplished by construction of a genomic DNA library in a bacteriophage ;~ vector followed by screening of the library using the transgene as a probej 4-6) However, this approach has at least two drawbacks. First, the preparation and screening of the genomic library is tedious and time consuming. Second, genomic DNA may contain sequences or covalent modifications (e.g., methylation) that render recom- binant bacteriophage clones "unstable" in packaging reactions or in host bac- teria, thus precluding efficient cloning of certain regions.

Alternative approaches have util- ized polymerase chain reaction (PCR)- based methods to facilitate the isolation of DNA adjacent to a region of known sequence identity. 17-18t These PCR strategies can be categorized con- ceptually into three classes referred to as circular (or inverse) PCR, anchor PCR, and single-site PCR. In circular (or inverse) PCR,/7-91 DNA to be amplified is first digested with a restriction endonuclease, then ligated under conditions that favor recircularization. After nicking of the circular DNA, PCR is performed using primers orientat-

ed outward from a region of known sequence identity. With anchor PCR, t1°-16) amplification occurs between a known sequence within a DNA molecule and a specific DNA sequence ligated to its termini. The ligated sequence may be a double- stranded DNA linker, (1°-13) a homo- polynucleotide tract generated by terminal transferase,(14,1s) plasmid DNA, (16) or other substrates. For single- site PCR,(17,18) a double-stranded oligonucleotide adaptor is ligated to restriction enzyme-digested or blunt- ended DNA. The double-stranded adaptor is designed such that it contains within it a region of single- stranded DNA, either as 3 ' terminal (17) or internal "bubble" mismatches. (18) With either version, the specificity of the PCR is generated by the prerequisite synthesis of DNA complementary to the single-stranded region of the adaptor. The primer for the initial DNA synthesis reaction is an oligonucleotide that binds to a region of known sequence identity within the template DNA. The other primer used in the PCR recognizes only the newly synthesized complementary DNA as a target annealing sequence.

We have been interested in charac- terizing the recessive sys mutation in a line of transgenic mice. (6) Previously, we isolated one flank of the single site of transgene integration in this line of mice by bacteriophage cloning.(6) However, despite screening of four in- dependent bacteriophage libraries con- structed with restriction endonuclease-

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digested, size-selected transgenic genomic DNA, we failed to isolate a clone conta in ing the second flank.

Therefore, we at tempted to isolate the second flank using a simplification of a single-site PCR technique (17) and compared this directly to an anchor- PCR technique.(16) Although both protocols were successful, the specificity of the simplified single-site PCR ap- peared greater than that of anchor PCR. In addit ion to this enhanced specificity, the simplified single-site PCR has advantages over previous single-site PCR methods in that it util- izes a single-stranded oligonucleotide, not double-stranded adaptors or splints, and that the oligonucleotide does not require kinasing prior to use. We describe here the modified technique and its application to the isolation of the transgenic flanking genomic DNA. This method facilitates the rapid isolation of genomic sequences flanking transgene integration sites and any other region of known sequence identity and in particular may assist in the isolation of those m a m m a l i a n DNA sequences that are recalcitrant to c loning in certain bacterial hosts.

METHODS DNA Restriction and Modification DNAs were treated with restriction endonucleases and modif icat ion enzymes as described by the manufac- turer (CIP, Boehringer Mannhe im; SstI, Bethesda Research Laboratories, Ampli- Taq, Perkin-Elmer Cetus; T4 DNA ligase and all other enzymes, Pharmacia LKB). The DNA ligation buffer used was that described by Lathe et al. (]9)

DNA Isolation and Synthesis Genomic and plasmid DNAs were isolated as described. (2°) Oligonucleotides were synthesized using an Applied Biosystems 380B DNA synthesizer. Fol- lowing deprotection and lyophiliza- tion, oligonucleotides were resuspended in HPLC-grade water and used directly. Genomic DNA from sys homozygous mice was digested with Sstl and then size-fractionated by electrophoresis through a Tris-acetate EDTA (TAE)-buffered agarose gel (SeaKem, FMC). (2°) Digestion with Sstl generates a 1.7-kb fragment with the desired transgenic genomic flank. Agarose

slices that contained DNA fragments of -1 .4-2.0 kb were generated and the DNA was recovered by sp inning through glass wool, (21) followed by precipitation at room temperature in the presence of 2.5 M a m m o n i u m acetate and 2.5 volumes of ethanol. A Southern analysis was performed to determine which fractions contained the desired DNA fragment.

Preparation of Single-site PCR Template Fifty nanograms of SstI-digested genomic DNA was mixed with the "ligation" oligonucleotide (see Fig. 1) in a 103-to-1 molar ratio (oligonucleotides to genomic DNA termini) in a final volume of 10 ,l . The mixture was heated to 65°C for 15 min (to dissociate any annealed genomic fragments) and then placed directly on ice. Three (Weiss) units of T4 DNA ligase were added and the reaction incubated

at 15°C overnight. After raising the reaction volume to 50 ,1 the reaction was heated to 65°C for 15 rain to inactivate the ligase and to dissociate unligated oligonucleotides and then was run through a Sephadex G 50-80 spin co lumn to remove the unligated oligonucleotides.

P r e p a r a t i o n o f A n c h o r PCR T e m p l a t e

Ten micrograms of pTZ19r was digested with SstI and the 5 ' termini dephosphorylated with CIP. Following phenol extraction and ethanol precipitation, (2°) the DNA was resuspended at a concentrat ion of 50 ~g/ml. Bacterial t ransformation was used to determine the efficiency of vector digestion and dephosphorylat ion. Fifty nanograms of size-selected or total SstI-digested genomic DNA was ligated to the vector in a 1-to-5 molar ratio (insert to vector) to maximize the proportion of insert

- - : : ~ S6t I digested genomic material (size fractionated)

"Internal" oligo, with sequence homology to transgene

"Ligation" oligo, with 3' end having complementarity to SstI 3' overhang

"External" oligo. Same as "ligation"oligo, except 4 base anneal seq is omitted

Anneal "ligation"oligo to SstI digested material

Ligate "ligation"oligo to SstI digested material. Remove excess linkers by passing material down G50-80 column.

. . . . Denature DNA, inactivate ligase at same time ~ (In thermal cycler).

. . . . .... Add Taq polymerase, anneal "internar'oligo _., ~ primer, perform primer extension (first cycle).

Now internal and external oligos have binding 5 - sites - perform PCR (second and subsequent

~ cycles)

FIGURE 1 0 l i g o ligation PCR strategy. The boxed section illustrates the components of the reaction. The individual steps of the reaction are illustrated in steps 1-5.

130 PCR Methods and Applications




ligated to vector. The ligation was performed overnight at room temperature (27°C) in a final volume of 10 ~1 using three (Weiss) units of T4 DNA ligase. Before initiating the PCR, the volume was raised to 50 , l and the reaction was heated to 95°C for 30 rain to inactivate the ligase and to nick the circular template./7)

PCR Amplification PCR was performed using the recommended Cetus buffer system [lx buffer = 50 mM KC1, 1.5 mM MgCI2, 10 mM Tris-HC1 (pH 8.3) at 25°C, 0.01% wt/vol gelatin] in an Ericomp twin- block thermal cycler. Oligonucleotides were used at 500 BM each and deoxynucleotides (dNTPs) at 250 jxM each. Five units of AmpliTaq (Perkin- Elmer Cetus) were used in each 100-Bl reaction. Total template DNA con- centrations in these reactions varied from 250 ng/ml for oligonucleotide ligation to 1 Bg/ml for vector ligation. Ten percent (5 B1) of each template was combined with buffer, dNTPs, and the "internal" (specific for the transgenic DNA) and "external" oligonucleotide primers; the mixture was heated to 95°C in the thermal cycler for 10 rain to denature the DNA and to inactivate any remaining ligase activity. Follow- ing the addition of Taq polymerase, the thermal cycler was taken off "hold" and 38 rounds of PCR performed using the following conditions: anneal 58°C, 5 sec; polymerize, 72oc, 2 rain 30 sec; denature, 95°C, 30 sec.

PCR Product Recovery and Subcloning PCR products were recovered by ethanol precipitation. Following digestion of the products with ap- propriate enzymes, they were subjected to agarose gel electrophoresis, excised from the agarose, and purified by spin-

ning through glass wool. (21) After a further ethanol precipitation, the products were quantified, subcloned into pTZ19r phagemid vector (Pharmacia), and screened using standard techniques. (20)

Southern Analysis Southern analysis on size-selected and total genomic DNA was performed as described.(20)

Oligonucleotide Primer Sequences The " internal" o l igonucleot ide was designed f rom the sequence of the SV40 genome that is present at the 3 ' end of the RSV lacZ transgene. (6) It has 24 bases of homology to SV40 (nucleotide numbers 2295-2319) and a HindIII site at the 5 ' end (plus 4 bases of "buffer" DNA to increase the efficiency of digestion of subsequent PCR products by HindIII). It has the sequence 5 ' -ATGCAAGCTTGTAAACAGCCCAC AAATGTCAACA-3 ' The "ligation" oligonucleotide, whose 3 ' end has complementarity to an SstI 3 ' overhang, contains a BamHI recognition site at its 5 ' end, and it has the sequence 5 ' -GGGATCCTGATGCAGTCA GTGCACTACGACAGCT-3 ' The "external" oligonucleotide is identical to the "ligation" oligonucleotide, except that the SstI annealing sequence (AGCT) is absent. The oligonucleotide used to amplify sequences cloned into pTZ19r was derived from the poly- linker and has the sequence 5 ' - GCCTGCAGGTCGACTCTAGAGGATC- 3 '

RESULTS A Simplified Single-site PCR We have s impl i f ied a single-site PCR strategy 117~ and have used this protocol to isolate genomic DNA flanking the integration site of a transgene complex in the sys family of transgenic mice. (6) The protocol is outlined in Figure 1. Initially, genomic DNA is digested with a restriction enzyme that generates a 3 ' overhang. Next, the digested DNA is annealed and ligated to a single- stranded oligonucleotide that has complementarity to the 3 ' overhang. This generates double-stranded templates with 3 ' recessed ends. Following removal of unligated oligonucleotides, the genomic DNA is denatured and an-

nealed to an "internal" oligonucleotide whose target sequence is within the transgene. Next, a primer extension reaction is performed, which termi- nates after synthesis of DNA complementary to the first, ligated, oligonucleotide. Finally, PCR is performed using a primer specific for the transgene and an "external" oligonucleotide that is identical to the "ligation" oligonucleotide but lacks the region of complementarity to the restriction en-

zyme digestion site 3 ' overhang. As with other single-site PCR strategies, the specificity of this reaction is generated by the "internal" oligonucleotide- mediated primer extension, which syn- thesizes the template DNA for the other primer in the PCR. The "external" primer should generate no amplification products until DNA complementary to the "ligation" oligonucleotide is synthesized by primer extension from the "internal" oligonucleotide.

Comparison of Simplified Single- site PCR and Anchor PCR in Isolating Specific Genomic DNA Sequences This simplified single-site PCR technique was compared directly to an anchor-PCR method (16) in their utility to isolate genomic DNA flanking a transgenic integration site. SstI- digested genomic DNA from homozygous sys transgenic mice was ligated either to the "ligation" oligonucleotide (see Methods) to generate a single-site PCR template or to pTZ19r to generate an anchor-PCR template. For each strategy, three different genomic DNAs were used: (1) size-fractionated genomic DNA that was positive for the transgenic flank, (2) size-fractionated material that was negative for the transgenic insert (as a negative con- trol), or (3) unfractionated, total genomic DNA. Successful PCR amplification is expected to generate a product of 1.4 kb, this being the distance from the "internal" oligonucleotide annealing site to the SstI genomic site.

Although both strategies resulted in successful amplification of the genomic sequences flanking the transgene (see Fig. 2), use of the size-fractionated genomic DNA with the simplified single-site PCR (lane 1) generated an amplification product of a more

specific nature than the equivalent anchor PCR (lane 4). The 1.4-kb species is the predominant reaction product in the single-site PCR; however, there are at least six other species present in the product from the anchor PCR.

With either protocol, the use of total digested genomic DNA as the PCR template failed to generate the specificity achieved with the size-fractionated genomic template. However, again, the simplified single-site PCR strategy suc- ceeded in generating the desired flank-

PCR Methods and Applications 131




ii!!iil !i i

1.4kb

m 1 2 3 4 5 6 7 8 9 10

FIGURE 2 Analysis of PCR reaction products. Ten percent of each PCR was run through a 0.7% TAE agarose minigel, stained with ethidium bromide, and photographed on a UV transilluminator. The table below gives information regarding the nature of the genomic DNA and whether the DNA was ligated to the ligation oligonucleotide (O), to pTZl9r vector DNA (V), or unligated (U). "Oligonucleotides" refers to the oligonucleotides used to perform the initial primer extension and to generate the subsequent PCR. (Lanes M) Markers, 1-kb ladder (Bethesda Research Laboratories). The arrow indicates the position of the predicted amplification product at 1.4 kb.

Lane Genomic DNA, Sstl-digested Ligated to Oligonucleotides

1 size-fractionated, positive for flank O 2 size-fractionated, negative for flank O 3 total O 4 size-fractionated, positive for flank V 5 size-fractionated, negative for flank V 6 total V 7 no genomic DNA, vector only V 8 total U 9 total U

10 total U 11 total U 12 total U

internal and external internal and external internal and external internal and vector internal and vector internal and vector internal and vector internal and external internal only external only vector only internal and vector

ing genomic material in appreciable quantit ies despite a background "smear" of nonspecific amplification products (see 1.4-kb product in Fig. 2, lane 3) whereas the equivalent anchor- PCR reaction could only generate the genomic flank as a minor amplifica- t ion product (Fig. 2, lane 6). Thus, with either size-selected or total digested genomic DNA as the template, the simplified single-site PCR generated the desired genomic flanking material with greater specificity than the equivalent anchor-PCR reaction.

We have not a t tempted to determine the nature of the additional, nonspecific PCR products tha t were generated. They may arise from

amplification of the other copies of the RSV-LacZ transgene, as this family has approximately 20 copies of the transgene integrated at a single-site and there are two SstI sites within each transgene.

Size-selected genomic DNA lacking t ransgene/f lanking material did not give an amplification product of 1.4 kb (Fig. 2, lanes 2 and 5). In addition, reactions in which the "ligation" oligonucleotide was not linked to the genomic DNA did not yield an amplification product (Fig. 2, lanes 8-12).

Following an ethanol precipitation, the remaining reactions from lanes 1 and 4 were digested with restriction enzymes whose target sequences had

been designed into the oligonucleotides. The single-site PCR material was digested with BamHI and HindlII, and the anchor-PCR material was digested with HindlII and Sail. The products were subjected to agarose gel electrophoresis, and the 1.4-kb fragment from each reaction was cloned following gel purification. (21

Confirmation of Identity of Amplified DNA as Transgenic Integration Site To confirm the identity of the cloned product as genomic material f lanking the transgenic integration site, an RFLP analysis was performed. The results are illustrated in Figure 3. The cloned PCR product was radiolabeled and hybridized to Sstl- or EcoRl-digested genomic DNA from nontransgenic, hemizygous and homozygous sys mice (Fig. 3). The Southern hybridization patterns confirm the identity of the amplified material as genomic DNA flanking the transgene integration site. For SstI- digested genomic DNAs, the probe detects a 1.85-kb band associated with the nontransgenic allele and a 1.7-kb band associated with the transgenic allele. Similarly, for EcoRl-digested DNAs, the probe detects a 2.2-kb band for the nontransgenic allele and a 6.4-kb band for the transgenic allele. The probe also hybridizes weakly to a 2.35-kb non- polymorphic band in the EcoRI digests. Identical results were obtained using products from the anchor PCR (data not shown).

DISCUSSION We have used a simplified single-site PCR technique (see Fig. 1) to isolate genomic DNA sequences flanking the transgenic integration site in a line of transgenic mice. The flanking material had previously proven refractory to isolation in Escherichia coli. In this regard, PCR-mediated isolation has dual advantages over more conven- tional cloning strategies. It is rapid and is unaffected by DNA modifications (e.g., methylat ion) that can interfere with efficient cloning.

Several PCR-based strategies have been described that facilitate the isolation of DNA adjacent to regions of known sequence identity. (7-18) Of these strategies, for isolation of flanking sequences, single-site PCR has the





SstI EcoRI

1.85 kB 1.70

6.4kb

kB

PCR prOduct

EcoRI

L a c Z p o l y A t ..... OVE3A transgene l integration site

t t, 1

SstI , 1 . 7 k b ss t I EcoI~

-~ .... 6 . 4 k b

FIGURE 3 Restriction fragment length polymorphism (RFLF) analysis of wild-type and sys al- leles using a cloned PCR product as a probe. Genomic DNAs from wild-type, heterozygous, and homozygous sys mice were digested with EcoRI or SstI, subjected to electrophoresis through a TAE agarose gel, and transferred to nylon membranes. These were hybridized with the radiolabeled cloned 1.4-kb PCR product, as illustrated by the schematic. An RFLP is detected for both the EcoRl- and Sstl.digested material. There is low-copy repetitive DNA that is detected with either probe, as a smear with Sstl-digested DNA or resolved by EcoRl digestion as a 2.35-kb band. The lower schematic illustrates the 3 ' end of the transgene complex including the location of Sstl and EcoRI restriction sites in the transgene and the flanking genomic DNA sequences. The position of the "internal" oligonucleotide used to perform the initial primer extension is indicated by P.

potential to generate highest product specificity.(17,18) This specificity is achieved by performing a primer extension reaction from a specific site wi th in a DNA fragment that results in synthesis of DNA complementa ry to a single-stranded region attached to the end of the fragment. It is this specific synthesis that generates the target sequence for a second oligonucleotide in a subsequent PCR.

In previously reported single-site PCR strategies, ~17,18) the single-stranded region has been produced by use of duplex adaptor DNA molecules that

are ligated to the ends of 5 ' overhang, restriction-digested,(17) or blunt- ended (18) template DNA. The adaptors contain a region of mismatch either at the 3 ' end of one molecule (resulting in the formation of a "tail ''~17)) or wi th in the duplex itself (resulting in the formation of a "bubble" c18)). The presence of this tail or bubble prevents copying of the single-stranded region of the duplex adaptor. Only in the case where DNA synthesis is pr imed wi th in the denatured template will successful copying of the single-stranded region occur.

Vital to the success of these single- site PCR strategies is the efficient kinasing of the oligonucleotides used to form the adaptor molecules. Failure to ligate the strand of the adaptor that in- hibits synthesis of DNA complementary to the single-stranded region of the adaptor will produce template molecules with a 3 ' recessed end. Sub- sequent addit ion of Taq polymerase will result in the synthesis of primer b inding sites at both ends of all molecules which could potential ly reduce the specificity of a subsequent PCR. However, this problem can be ob- viated by prior denaturat ion of the DNA. it follows that no advantage ac- crues from use of a double-stranded splint or bubble duplex except the ability to ligate these classes of molecules onto DNA digested with restriction enzymes that generate 5 ' overhangs or blunt-ended molecules.

We have simplified these single-site PCR methods to utilize a single- stranded oligonucleotide instead of the more cumbersome double-stranded adaptors or splints. This has dual advantages of reduced expense for oligonucleotide synthesis and no require- ment for kinasing prior to use, because the 5 ' end does not participate in the ligation reaction. Although this single- site PCR strategy requires the digested template DNA to have a 3 ' overhang, there are several commercial ly available restriction endonucleases that generate nonredundant , four-base, 3 ' overhangs, e.g., AatlI, ApaI, HaelI, Kpnl, NlallI, NsiI, NspHl, PstI, SstI, and Sphl. Oligonucleotides can be designed that are complementary to the 3 ' overhang of the particular restriction enzyme used.

We performed an experimental comparison of the relative abilities of the simplified single-site PCR and an anchor-PCR method (16) to amplify genomic material f lanking a transgene integration site in mouse genomic DNA. Enhanced product specificity (the relative ratio of specific to nonspecific PCR product) was observed ex- per imental ly using size-selected genomic DNA as the template when we compared the simplified single-site PCR to the anchor-PCR method (in the latter, specific oligonucleotide pr iming from an internal sequence is not re- quired to generate the second oligonu-





cleotide binding site). This ability of the simplified single-site reaction to generate a more specific PCR product was also observed when total genomic DNA was used as template, a l though these reaction specificities were lower than those achieved with size-selected, f lank-containing genomic DNA. Over- all, the simplified single-site PCR ap- pears superior to the anchor PCR- method. 116) Although a direct comparison with other single-site PCR strategies is difficult to make, the simplified single-site PCR technique does appear to have greater sensitivity and specificity compared to one of its predecessors.i 17)

In performing the primer extension step of the simplified single-site PCR, it is impor tan t to ensure thorough denaturat ion of the template. In practice, this can be achieved by placing the thermal cycler on "hold" at 95°C while performing the initial denaturat ion and, following addit ion of Taq polymerase with the Eppendorf tube in the block, taking the machine off "hold" to start the first cycle. Thorough denaturat ion will eliminate the synthesis of DNA complementary to the ligated oligonucleotide via extension of 3 ' recessed ends of nondena tured template and prohibit the formation of er- roneous primer extension products. During the anneal ing step of the first cycle, the complexi ty of the genomic mixture is sufficient to preclude synthesis of DNA complementary to the "ligation" oligonucleotide that could occur through reassociation of genomic DNA strands.

Also, it is vital that the highest pos- sible anneal ing temperature be used to ensure reaction specificity. In our expe- rience, using primers with 25 bases of homology, 45-55% GC content, and the Cetus-recommended buffer system, specificity of anneal ing was achieved using temperatures between 55°C and 60°C. In theory, with this anneal ing temperature range, it should be pos- sible to simplify the reaction components further. As the "ligation" oligonucleotide has only 4 bases of homology at its 3 ' end to the 3 ' ends of the digested genomic DNA, this oligonucleotide could be used to replace the "external" oligonucleotide in the PCR. However, experimentally, all a t tempts to replace the "external"

oligonucleotide in this manner have resulted in drastic loss of specificity of subsequent PCR products (data not shown).

Due to the presence of more than one copy of the transgene in the particular line of mice analyzed, to achieve the highest reaction specificity, it was necessary to gel-purify digested genomic DNA containing the ap- propriate target sequence prior to liga- t ing the "ligation" oligonucleotide and performing the PCR. (However, amplification of the desired genomic material was achieved, albeit with lower specificity using total digested genomic DNA as the template.) In cases where additional restriction map- ping data is available, the specificity of amplification might be enhanced by digesting the template DNA with additional enzyme(s). The additional enzymes would be chosen to restrict annealing of the "ligation" oligonucleotide to a subset of the digested DNA thereby prevent ing amplification of internal copies of the transgene.

The main advantages of this single- site PCR technique are its simplicity, the minimal reagent requirements, and its ability to generate the desired product even from templates of high com- plexity such as total digested genomic DNA. It may prove useful not only for this, but for additional problems such as definition of in t ron-exon boundaries using cDNA sequence information and isolation of retroviral integration sites.

ACKNOWLEDGMENTS

We thank Stephen Cohen and Richard Gibbs for helpful comments on the manuscript . G.R.M. is a Research Asso- ciate and P.A.O. an assistant investiga- tor of the Howard Hughes Medical In- stitute. This work was also supported by the National Institutes of Health (HD 25340).

REFERENCES 1. Palmiter, R.D. and R.L. Brinster. 1986.

Germ line transformation of mice. Annu. Rev. Genet. 20: 465-499.

2. Gridley, T., P. Soriano, and R. Jaenisch. 1987. Insertional mutage- nesis in mice. Trends Genet. 3: 162-166.

3. Costantini, F., G. Radice, J.L. Lee, K.K.

Chada, W. Perry, and H.J. Son. 1989. Insertional mutations in transgenic mice. Progr. Nucleic Acids Res. Mol.

Biol. 36:159-169. 4. Shawlot, W., M.J. Siciliano, R.L.

Stallings, and P.A. Overbeek. 1989. lnsertional inactivation of the downless gene in a family of transgenic mice. Moi. Biol. Med. 6: 299-307.

5. Xang, X., K.F. Benson, and K. Chada. 1990. Mini-mouse: Disruption of the pygmy locus in a transgenic insertional mutant. Science 247: 967-969.

6. MacGregor, G.R., L.D. Russell, M.A.E. Van Beek, G.R. Hanten, M.J. Kovak, C.A. Kozak, M.L. Meistrich, and P.A. Overbeek. 1990. Symplastic sperma- tids (sys): A recessive insertional mutation in mice causing a defect in spermatogenesis. Proc. Natl. Acad. Sci.

87: 5016-5020. 7. Triglia, T., M.G. Peterson, and D.J.

Kemp. 1988. A procedure for in vitro

amplification of DNA segments that lie outside the boundaries of known sequence. Nucleic Acids Res. 16: 8186.

8. Ochman, H., A.S. Gerber, and D.L. Hartl. 1988. Genetic applications of an inverse polymerase chain reaction. Genetics 120: 621-623.

9. Silver, J. and V. Keerikatte. 1989. Novel use of polymerase chain reaction to amplify cellular DNA adjacent to an integrated provirus. J. Virol. 63: 1924-1928.

10. Meuller, P.R. and B. Wold. 1989. In vivo footprinting of a muscle specific enhancer by ligation mediated PCR. Science 246: 780-786.

l l. Pfeifer, G.P., S.D. Steigerwald, P.R. Mueller, B. Wold, and A.D. Riggs. 1989. Genomic sequencing and methylation analysis by ligation mediated PCR. Science 246: 810-813.

12. Rosenthai, A. and D.S. Jones. 1990. Genomic walking and sequencing by oligo-cassette mediated polymerase chain reaction. Nucleic Acids Res. 18: 3095-3096.

13. Fors, L., R.A. Saavedra, and L. Hood. 1990. Cloning of the shark PO promoter using a genomic walking technique based on the polymerase chain reaction. Nucleic Acids Res. 18: 2793-2799.

14. Loh, E.Y., J.F. Elliot, S. Cwerla, L.L. Lanier, and M.M. Davis. 1989. Poly- merase chain reaction with single sided specificity: Analysis of T-cell





receptor delta chain. Science 243: 217-220.

15. Frohman, M.A., M.K. Dush, and G.R. Martin. 1988. Rapid production of full length cDNA's from rare transcripts using single gene specific oligonucleotide primer. Proc. Natl. Acad. Sci. 85" 8998-9002.

16. Shyamala, V. and G.F.-L. Ames. 1989. Genome walking by single-specific- primer polymerase chain reaction: SSP-PCR. Gene 84: 1-8.

17. Roux, K.H. and P. Dhanarajan. 1990. A strategy for single site PCR amplification of dsDNA: Primer digested cloned or genomic DNA from an anchor modified restriction site and a short internal sequence. Biotechniques 8: 48-57.

18. Riley, J., R. Butler, D. Ogilvie, R. Finniear, D. Jenner, S. Powell, R. Anand, J.C. Smith, and A.F. Markham. 1990. A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones. Nucleic Acids Res. 18; 2887-2890.

19. Lathe, R., S. Skory, and M.-P. Kieny. 1984. Inserting new restriction sites by linker tailing. Focus 6:4, 1-6.

20. Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular cloning: A laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

21. Heery, D.M., F. Gannon, and R. Powell. 1990. A simple method for subcloning DNA fragments from gel slices. Trends. Genet. 6:173.

Received May 10, 1991; accepted in revised form August 26, 1991.





10.1101/gr.1.2.129Access the most recent version at doi:1991 1: 129-135 Genome Res.

G R MacGregor and P A Overbeek DNA sequences flanking a transgene integration site.Use of a simplified single-site PCR to facilitate cloning of genomic

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